Study On Genetic Modification Of Industrial Avermectin Producer Strains Streptomyces Avermitilis

Avermectin,the main secondary metabolites produced by Streptomyces avermitilis,and its derivatives with structural similarity are a series of 16-membered macrocyclic anthelmintic antibiotics.Because of the high efficiency,low toxicity,broad-spectrum insecticidal activity,they are widely utilized in agriculture,medicine and animal husbandry.For the purpose of production improvement,many trials have been made by genetic engineering in addition to traditional strains mutation and screening,but so far it is difficult to further rationally improve the production capacity of industrial overproducers.Most modification strategy does not work well for the two reasons:firstly,the molecular genetic basis of industrial overproducers is complicated and most of target sequences aimed by the genetic engineering strategy have been undergone tremendous changes;secondly,it is difficult to make some multistep manipulation to industrial strains and evaluate the actual effect of the modification strategy,on account of the propagating instability caused by strain degradation.In this study,some key parameters optimization were made in conjucation of the industrial strains preserved in our laboratory on the basis of classical genetic approach,including MgCl2 concentration,time and temperature of heat-shock,pre-germination time,ratio of donor to recipient and the timing of antibiotics overlaying.After optimization of all these conditions,the efficiency of conjugation was increased by 3.4 times finally.Deletion mutants of different PKS biosynthesis cluster were performed and constructed by homologous recombination under the optimized condition and finally seven PKS biosynthesis clusters(pks2,pks5,olm,pte,pks9,rpp,pks11)were knocked out successfully.These deletion mutants were named AV002,AV005,AV006,AV007,AV009,AV010 and AV011 respectively.Among them,AV002 and AV005 no longer produced avermectin.Besides,the avermectin production in AV011 decreased by 18%and AV009 showed no remarkable change on avermectin production compared with the parent strain.On the contrary,the avermectin production in AV006,AV007 and AV010 were enhanced to varying degrees.Fortunately,the most significant improvement(nearly 25%)of avermectin production was found in AV010,in which the rpp biosynthesis cluster was deleted completely.The avermectin production was enhanced to 1262 mg l-1 from 1024 mg l-1.Based on bioinformatics analysis we found that the rpp biosynthetic gene cluster(sav7130-7131)in Streptomyces avermitilis contained a type ? PKS and a cytochrome P450 and was reportedly involved in producing a diffusible brown pigment.Since the same precursor malonyl-CoA was used as substrate for the type ? PKSs and type ? PKSs,it could be speculated that between rpp and avermectin biosynthetic clusters there might be a competition for precursor supply.After constructions of the rpp deletion mutant and the complementary strain,we confirmed that the increased avermectin production in AV010 was solely due to the deletion of rpp biosynthetic gene cluster without any detectable effect on the cell growth.While,the production of an industrial overproducer increased from 3582 mg l-1 to 4450 mg l-1 in the same way.Transcriptional analysis suggested that the deletion of rpp gene cluster in the industrial overproducer AV-HP stimulated transcription of aveR(5 folds up-regulated),leading to increased transcription of structure genes,aveA1(26 folds up-regulated),aveA2(11 folds up-regulated),aveA3(7 folds up-regulated),aveA4(15 folds up-regulated)and a consequent increase in avermectin production.Two-component system is an important regulatory mechanism of secondary metabolite biosynthesis in Streptomyces.Based on genomic and phylogenetic analysis,we found the two-component system sav931/932 adjacent to avermectin biosynthesis cluster might have some effects on regulation of avermectin biosynthesis,because of the 53%sequence similarity between SAV931 and AbrA2,the negative regulatory factor of RED in Streptomyces coelicolor.Deletion of sav931/932 in Streptomyces avermitilis ATCC31267 resulted in the increase of avermectin production by more than 25%,and faster mycelium growth in the prophase of fermentation.RT-PCR showed that the transcription level of the structure genes,aveA1,aveA3 and the regulatory gene aveR involved in avermectin biosynthetic pathway were up-regulated by 70%,30%and 30%,respectively.These results suggested that two-component system sav931/932 inhibited avermectin biosynthesis to some extent.These results may be helpful to better understand the regulatory mechanism of secondary metabolite and improve avermectin production in Streptomyces avermitilis industrial strains.